ABC Efflux Transporters and the Circuitry of miRNAs: Kinetics of Expression in Cancer Drug Resistance.

Cancer drug resistance (CDR) is a major problem in therapeutic failure. Over 90% of patients with metastatic cancer present CDR. Several mechanisms underlie CDR, including the increased expression of efflux ABC transporters and epigenetic phenomena. Nevertheless, a topic that is not usually addressed is the mechanism underlying the loss of CDR once the challenge to these cells is withdrawn. A KCR cell line (doxorubicin-resistant, expressing ABCB1) was used to induce loss of resistance by withdrawing doxorubicin in culture medium. ABCB1 activity was analysed by fluorescence microscopy and flow cytometry through substrate (DiOC2) retention assays. The expression of 1008 microRNAs was assessed before and after doxorubicin withdrawal. After 16 weeks of doxorubicin withdrawal, a decrease of ABCB1 activity and expression occurred. Moreover, we determined a signature of 23 microRNAs, 13 underexpressed and 10 overexpressed, as a tool to assess loss of resistance. Through pathway enrichment analysis, "Pathways in cancer", "Proteoglycans in cancer" and "ECM-receptor interaction" were identified as relevant in the loss of CDR. Taken together, the data reinforce the assumption that ABCB1 plays a major role in the kinetics of CDR, and their levels of expression are in the dependence of the circuitry of cell miRNAs.

The Complex Dynamic of Phase I Drug Metabolism in the Early Stages of Doxorubicin Resistance in Breast Cancer Cells.

The altered activity of drug metabolism enzymes (DMEs) is a hallmark of chemotherapy resistance. Cytochrome P450s (CYPs), mainly CYP3A4, and several oxidoreductases are responsible for Phase I metabolism of doxorubicin (DOX), an anthracycline widely used in breast cancer (BC) treatment. This study aimed to investigate the role of Phase I DMEs involved in the first stages of acquisition of DOX-resistance in BC cells. For this purpose, the expression of 92 DME genes and specific CYP-complex enzymes activities were assessed in either sensitive (MCF-7 parental cells; MCF-7/DOXS) or DOX-resistant (MCF-7/DOXR) cells. The DMEs genes detected to be significantly differentially expressed in MCF-7/DOXR cells (12 CYPs and eight oxidoreductases) were indicated previously to be involved in tumor progression and/or chemotherapy response. The analysis of CYP-mediated activities suggests a putative enhanced CYP3A4-dependent metabolism in MCF-7/DOXR cells. A discrepancy was observed between CYP-enzyme activities and their corresponding levels of mRNA transcripts. This is indicative that the phenotype of DMEs is not linearly correlated with transcription induction responses, confirming the multifactorial complexity of this mechanism. Our results pinpoint the potential role of specific CYPs and oxidoreductases involved in the metabolism of drugs, retinoic and arachidonic acids, in the mechanisms of chemo-resistance to DOX and carcinogenesis of BC.

HBM4EU Chromates Study-Genotoxicity and Oxidative Stress Biomarkers in Workers Exposed to Hexavalent Chromium.

A study was conducted within the European Human Biomonitoring Initiative (HBM4EU) to characterize occupational exposure to Cr(VI). Herein we present the results of biomarkers of genotoxicity and oxidative stress, including micronucleus analysis in lymphocytes and reticulocytes, the comet assay in whole blood, and malondialdehyde and 8-oxo-2'-deoxyguanosine in urine. Workers from several Cr(VI)-related industrial activities and controls from industrial (within company) and non-industrial (outwith company) environments were included. The significantly increased genotoxicity (p = 0.03 for MN in lymphocytes and reticulocytes; p < 0.001 for comet assay data) and oxidative stress levels (p = 0.007 and p < 0.001 for MDA and 8-OHdG levels in pre-shift urine samples, respectively) that were detected in the exposed workers over the outwith company controls suggest that Cr(VI) exposure might still represent a health risk, particularly, for chrome painters and electrolytic bath platers, despite the low Cr exposure. The within-company controls displayed DNA and chromosomal damage levels that were comparable to those of the exposed group, highlighting the relevance of considering all industry workers as potentially exposed. The use of effect biomarkers proved their capacity to detect the early biological effects from low Cr(VI) exposure, and to contribute to identifying subgroups that are at higher risk. Overall, this study reinforces the need for further re-evaluation of the occupational exposure limit and better application of protection measures. However, it also raised some additional questions and unexplained inconsistencies that need follow-up studies to be clarified.

The role of CCNH Val270Ala (rs2230641) and other nucleotide excision repair polymorphisms in individual susceptibility to well-differentiated thyroid cancer.

Well-differentiated thyroid cancer (DTC) is the most common form of thyroid cancer (TC); however, with the exception of radiation exposure, its etiology remains largely unknown. Several single nucleotide polymorphisms (SNPs) have previously been implicated in DTC risk. Nucleotide excision repair (NER) polymorphisms, despite having been associated with cancer risk at other locations, have received little attention in the context of thyroid carcinogenesis. In order to evaluate the role of NER pathway SNPs in DTC susceptibility, we performed a case-control study in 106 Caucasian Portuguese DTC patients and 212 matched controls. rs2230641 (CCNH), rs2972388 (CDK7), rs1805329 (RAD23B), rs3212986 (ERCC1), rs1800067 (ERCC4), rs17655, rs2227869 (ERCC5), rs4253211 and rs2228529 (ERCC6) were genotyped using TaqMan® methodology, while conventional PCR-RFLP was employed for rs2228000 and rs2228001 (XPC). When considering all DTC cases, only rs2230641 (CCNH) was associated with DTC risk; a consistent increase in overall DTC risk was observed for both the heterozygous genotype (OR=1.89, 95% CI=1.14-3.14) and the variant allele carriers (OR=1.79, 95% CI=1.09-2.93). Histological stratification analysis confirmed an identical effect on follicular TC (OR=2.72, 95% CI=1.19-6.22, for heterozygous; OR=2.44, 95% CI=1.07­5.55, for variant allele carriers). Considering papillary TC, the rs2228001 (XPC) variant genotype was associated with increased risk (OR=2.33, 95% CI=1.05-5.16), while a protective effect was observed for rs2227869 (ERCC5) (OR=0.26, 95% CI=0.08­0.90, for heterozygous; OR=0.25, 95% CI=0.07-0.86, for variant allele carriers). No further significant results were observed. Our results suggest that NER polymorphisms such as rs2230641 (CCNH) and, possibly, rs2227869 (ERCC5) and rs2228001 (XPC), may influence DTC susceptibility. However, larger studies are required to confirm these results.